Inhibition Test with compound 7575772. (Marshall's Top Scoring Compound)
Compound showed no signs of Inhibition. Orthovanadate did inhibit the YopH protein.
11/30/11
Inhibition Test with compound 5153298
Compound did not inhibit the protein. Orthovanadate did inhibit the YopH protein.
11/29/11
Vary Substrate (pNPP) Test
Different amounts of pnPP affected the absorbance of the YopH enzyme. The optimal volume of pNPP chosen was 16 uL.
11/28/11
Phosphatase Enzyme Test
Enzyme Works
Volume of Enzyme vs Absorbance graph. Optimal volume of enzyme was 30 uL or 60 nanograms.
Volume of Enzyme added vs Absorbance graph. Optimal volume of enzyme was 30 uL or 60 nanograms.
Week 12:
11/21
FLPC and Concentration.
Peak between 32-38 is the dimer sample of YopH. Peak between 40-45 is the monomer sample of YopH
The size of the YopH protein is 33512.8 daltons.
Week 11:
11/18/2011
Dried the gel in the biotech lab for 2 hours at 70 C.
11/15/2011
Ran all my samples on an SDS page gel. Gel shows that my protein sample might have some impurities.
Lane 1 contains NEB PageRuler Prestained Protein Ladder. Lane 2 contains cell lysate sample before induction. Lane 3 contains cell lysate sample after induction. Lane 4 contains the soluble fraction sample. Lane 5 contains the flow through sample. Lane 6 contains the wash sample. Lane 7 contains the elution 1 sample. Lane 8 contains the elution 2 sample. The YopH protein can be found in lanes 7 and 8. The two distinct bands in each lane represent the YopH protein. The higher bands represents dimer formation of the YopH protein. The lower bands represents YopH as a monomer.
Week 10:
11/09/11
Got rid of pellet and stored supernatant in frdige.
11/08/11
Attended Symposium at AT&T Conference center.
11/07/11
Submitted top 5 compounds to order. Top compound was 5153298
Week 9:
11/05/2011
Day 3 of Protein Expression. Stored pellet sample in -80 C freezer. Weight of Pellet = 2.78 g
11/04/2011
Day 2 of Protein Expression.
Picked 2 colonies to grow up from agarose plate containing BL21 (DE3) cells + YopH
11/03/11
Ran some virtual screen jobs against the ChemBridge-Diversit3d.sdf library. Had to make several conf files since the library was too big to screen.
11/01/11
Positive results from DNA sequencing. Image coming later.Began Day 1 of expression. Transformed plasmid into BL21 cells. Grew up positive colony from master plate too.
Sample 1 results. Foward Primer. Unmatched basepairs towards the end were compared to the reverse sequence.
Reverse Sequence of Sample 1.
Week 8:
10/27/2011
Nanodropped and sent 4 samples with plic primers to sequencing.
10/26/2011
Centrifuged colonies and miniprepped.
10/24/2011
Prepared master plate. picked 8 colonies to grow up.
10/23/11
Cohesive end generation of PCR Inserts and Accepting Vector. Anealing and transformation.
Week 7:
10/20/11
Submitted first run of the HF9 Library.
No colonies grew up on plate. Prepared more vector. Made more PCR product.
10/19/11
Cohesive End Generation on PCR insert and Accepting Vector and Anealing and Transformation and gel check on pNIC-Bsa vector simultaneously.
Lane 2: 1 kb ladder Lane 4: pNIC-Bsa4, Lane 6: pNIC-Bsa4. Both samples only contain one band. RE digest did not work.
10/16/11 Prepared pNIC-Bsa4 vector with BsaI.
Week 6:
10/14/2011
PCRsquared concentration.
Lane 2: 100 bp ladder Lane 3: Primary PCR Lane 4: Secondary PCR
YopH gene (921 bp) looks the correct size.
10/13/2011 Sequencing Results
10/10/2011
Sent 4 samples with for/rev primers to DNA sequencing
Week 5: 10/3/11
Completed Transformation on 10/07/11. DNA sequencing on Oct 10.
Plate A: contains DH5-a cells with pNIC-Bsa4 plasmid and possible YopH gene. Picked 8 colonies from this plate.
Concentration of PNic-BSA4 vector.
Transformation on 10/4/11
PCR squared sample. YopH Gene
Week 4: 9/26/11
Was not able to log in a lot of hours this week. Will make up for it.
1kb ladder with 3 different samples of cut pnic bsa4 vector
Week 3: 9/19/11
Prepared 3 more samples of Pnic Bsa4 vector. need to purify and gel check.
Prepared accepting PNic Bsa4 vector. Failed
Vector is cut in to places but the 2 dna fragments are about the same length which is bad.
Week 2: 9/12/11
Little work done in lab. Spent a lot of time working on research poster and at LSAMP conference.
Week 1:
J.C. - looks good. - Dr. B
3rd Try: Primary/Secondary PCR Results 9/7/11
2ndary PCR doesn't look too good. Ran PCR squared after. Might have to run again for a better band or use some of marshall's sample.
100 bp ladder, primary pcr, secondary pcr. One distinct band and one light band.
2nd Try: Primary/Secondary PCR Results 9/6/11
Used old primary pcr from the first try and made a second primary pcr. Diluted Custom primers. Used first primary pcr sample for the third try because it was similar to the primary pcr sample from the summer.
skip. 100 bp ladder, primary pcr, secondary pcr, primary pcr, secondary pcr. Secondary PCRs did not show up. Must of messed up on the pipetting part. :/
1st Try: Primary/Secondary PCR Results 9/1/11
1 kb ladder, secondary pcr, same sample of secondary pcr, primary pcr at the end. Contamination on secondary pcr. re did.
Summer 2011
Transformation Efficiency Lab
Plate with 1 ng of pGFP or 6.329 ul of dilution 3. 15 colonies
Plate with 5 ng of pGFP or 3.165 ul of dilution 2. 125 colonies
Restriction Enzyme Digest
Lane 1 (skip) Lane 2 (1 kb DNA ladder) Lane 3 (Uncut Plasmid) Lane 4 (EcoRI) Lane 5 (PvuII) Lane 6 (EcoRI + PvuII)
PGBR22 PCR
Lane 1 (Skipped) Lane 2 (100bp ladder) Lane 3 (Sample A) Lane 4 (Sample B) Lane 5 (Sample C) Lane 6 (Sample D) Lane 7 (100 bp ladder) Lane 8 (Sample A) Lane 9 (Sample B) Lane 10 (Sample C) Lane 11 (Sample D) Lane 12 (Skipped) Gel was shared. Lanes 1-6 Joey Lanes 7-12 JC
pGFP PCR
Lane 1 (Empty) Lane 2 (100bp ladder) Lane 3 (Sample 1 w VDS Primer) Lane 4 (Sample 2 w VDS Primer) Lane 5 (Sample 3 w VDS Primer) Lane 6 (Sample 4 with VDS primer) Lane 7 (Sample 5 w M13 Primer) Lane 8 (Sample 6 w M13 Primer) Lane 9 (Sample 7 w M13 Primer) Lane 10 (Sample 8 w M13 Primer)
pLIC PCR
Dropped the gel right after it finished running. I got the remains of it and place it in the UV Display machine. Only 100 bp ladder is visible. Still need to redo.
pNIC- Bsa4 (CA7) Target: HSCD00041026
1st Try. Gel was to thin, so the image would not show up on screen. Didn't work though.
Second try. Tried new temperature settings but still used the same other solutions, such as MgCl2.
Third try. Used the same temperature settings as before and used MgSO4 instead of MgCl2. Still no results.
Custom Primer Overlap Gel
Lane 1 (Empty) Lane 2 (100 bp DNA ladder) Lane 3 (Joey's Primary PCR) Lane 4 (Joey's Secondary PCR) Lane 5 (Larry's Primary PCR) Lane 6 (Larry's Secondary PCR) Lane 7 (JC's Primary PCR) Lane 8 (JC's Secondary PCR
JC's research
Week 13:
12/2/11
Inhibition Test with compound 7575772. (Marshall's Top Scoring Compound)11/30/11
Inhibition Test with compound 5153298
11/29/11
Vary Substrate (pNPP) Test
11/28/11
Phosphatase Enzyme TestEnzyme Works
Week 12:
11/21FLPC and Concentration.
The size of the YopH protein is 33512.8 daltons.
Week 11:
11/18/2011
Dried the gel in the biotech lab for 2 hours at 70 C.11/15/2011
Ran all my samples on an SDS page gel. Gel shows that my protein sample might have some impurities.Week 10:
11/09/11
Got rid of pellet and stored supernatant in frdige.11/08/11
Attended Symposium at AT&T Conference center.11/07/11
Submitted top 5 compounds to order. Top compound was 5153298Week 9:
11/05/2011
Day 3 of Protein Expression. Stored pellet sample in -80 C freezer. Weight of Pellet = 2.78 g11/04/2011
Day 2 of Protein Expression.11/03/11
Ran some virtual screen jobs against the ChemBridge-Diversit3d.sdf library. Had to make several conf files since the library was too big to screen.11/01/11
Positive results from DNA sequencing. Image coming later.Began Day 1 of expression. Transformed plasmid into BL21 cells. Grew up positive colony from master plate too.Week 8:
10/27/2011
Nanodropped and sent 4 samples with plic primers to sequencing.10/26/2011
Centrifuged colonies and miniprepped.10/24/2011
Prepared master plate. picked 8 colonies to grow up.10/23/11
Cohesive end generation of PCR Inserts and Accepting Vector. Anealing and transformation.Week 7:
10/20/11
Submitted first run of the HF9 Library.No colonies grew up on plate. Prepared more vector. Made more PCR product.
10/19/11
Cohesive End Generation on PCR insert and Accepting Vector and Anealing and Transformation and gel check on pNIC-Bsa vector simultaneously.10/16/11 Prepared pNIC-Bsa4 vector with BsaI.
Week 6:
10/14/2011
YopH gene (921 bp) looks the correct size.
10/13/2011 Sequencing Results
10/10/2011
Sent 4 samples with for/rev primers to DNA sequencingWeek 5: 10/3/11
Completed Transformation on 10/07/11. DNA sequencing on Oct 10.Transformation on 10/4/11
Week 4: 9/26/11
Was not able to log in a lot of hours this week. Will make up for it.Week 3: 9/19/11
Prepared 3 more samples of Pnic Bsa4 vector. need to purify and gel check.Prepared accepting PNic Bsa4 vector. Failed
Week 2: 9/12/11
Little work done in lab. Spent a lot of time working on research poster and at LSAMP conference.Week 1:
J.C. - looks good. - Dr. B3rd Try: Primary/Secondary PCR Results 9/7/11
2ndary PCR doesn't look too good. Ran PCR squared after. Might have to run again for a better band or use some of marshall's sample.2nd Try: Primary/Secondary PCR Results 9/6/11
Used old primary pcr from the first try and made a second primary pcr. Diluted Custom primers. Used first primary pcr sample for the third try because it was similar to the primary pcr sample from the summer.1st Try: Primary/Secondary PCR Results 9/1/11
Summer 2011
Transformation Efficiency Lab
Restriction Enzyme Digest
PGBR22 PCR
pGFP PCR
pLIC PCR
pNIC- Bsa4 (CA7) Target: HSCD00041026
1st Try. Gel was to thin, so the image would not show up on screen. Didn't work though.Custom Primer Overlap Gel